Study Of Aristolochic Acid Nephropathy And Its Toxicological Mechanism By UPLC-HDMS-based Metabolomics

Aristolochic acid nephropathy?AAN?is a common and rapidly progressive interstitial nephropathy which is caused by ingestion of Aristolochia herbal medications.The pathophysiology and molecular mechanisms of AAN which is not clear completely and the use of Aristolochia herbal drugs is restricted in the clinic.Metabolomics is an important method of system biology,which tried to comprehensively analyze changes of endogenous metabolites in organisms.Metabolomics was widely applied in the fields of disease diagnosis and drug toxicity evaluation.ObjectiveBased on the ultra performance liquid chromatography high-definition mass spectrometry?UPLC-HDMS?metabolomics and pharmacological methods,the changes of endogenous metabolites in different stages of rats with AAN were investigated systematically,and potential pathological mechanism was clarified in order to provide reference for the reasonable using of traditional Chinese medicine containing aristolochic acids.MethodsThe male Sprague-Dawley rats weighting 200 ± 10 g,were randomized to the AAN group?n = 30?and control group?n = 30?.The AAN group was fed with 20 mg/kg body weight/week of AAI in 0.5%NaHCO₃ solution by oral gavage for 12 weeks and observed for an additional 12 weeks.The control group was treated with the vehicle instead of AAI.After a time period of 0 week,4th week,8th week,12th week and 24th week,eight rats from each group were selected randomly and placed in metabolic cages to obtain 24 hr urine sample.Blood samples were obtained by carotid artery cannula at the same time kidneys were removed after processed for histological evaluation and metabolomics analyses.Results1.Urinary sample was analyzed by UPLC-HDMS-based metabolomics in AAN rats and sixteen,fourteen and fifteen biomarkers were identified at 4th week,8th week and 12th week respectively.The analysis of biochemical pathways indicated the perturbations of krebs cycle,gut microflora metabolism,amino acid metabolism,purine metabolism and bile acid metabolism are associated with AAN metabolic pathways.2.At 4th week,serum metabonomics results showed that the increase in taurochenodesoxycholic acid?TCDCA?and 12-ketodeoxycholic acid?12-KDCA?and the decrease in LysoPC?15:0?and docosahexaenoic acid?DHA?were observed in AAN rats,prior to detectable changes in conventional chemical markers and histological evidence of kidney injury;LysoPE?22:5?,indoxyl sulfate,uric acid and creatinine were found in the 12th week only,Thus they could be regarded as biomarkers of advanced AAN;LysoPE?20:2?,cholic acid?CA?,chenodeoxycholic acid?CDCA?,cystathionine sulfoxide and lysoPC?17:0?were significantly altered in AAN rats at weeks 4,8 and 12,therefor it is considered that these metabolites could potentially serve as biomarkers for progressive AAN.These altered metabolites were associated with metabolisms of lipids,amino acids and purine metabolism.3.Renal tissue metabonomics results demonstrated changes in lipids including PC,TG,PE,lysoPE and lysoPC were closely association with the disturbances of glycerophospholipid,glycerolipid,prenol lipid,sphingolipid metabolism,fatty acid oxidation,and bile acid biosynthesis.The study of pathological activity in the renal tissue suggested that kidney injury associated with activation of pro-inflammatory,pro-oxidant,and fibrotic pathways.ConclusionUPLC-HDMS-based metabolomics can be used to study the biochemical action mechanism of AAN,and the changes of different metabolites were detected in different stages of AAN.The mechanism of biochemical action in AAN was closely associated with the disorders of intestinal microbial metabolism,amino acid metabolism,purine metabolism,bile acid metabolism and lipid metabolism.